Starch is a complex polymer of glucosyl residues and is the major storage form for carbohydrates in higher plants. It is used to supply the needs of the plant for energy and biosynthesis when photosynthesis is not occurring. Starch accumulates in the chloroplasts of photosynthetic cells, as well as in amyloplasts present in storage organs such as seeds, fruit and tubers.
Starch is a mixture of two polysaccharides: amylose, which is a linear chain of glucosyl units linked by alpha-1,4-glycosidic bonds; and amylopectin, which is made up of many linear chains of alpha-1,4-polyglucans which are joined together by alpha-1,6 glycosidic bonds. The amylose/amylopectin ratio varies between plants and can affect the physical properties of starch, such as gelatinization temperature, retrogradation and viscosity.
Many plants with altered starch granules have been identified, and particular applications utilizing starch having these alterations have been recognized. Starch with no amylose (all amylopectin) is called “waxy” starch. Waxy starch, once gelatinized, is more rapidly digested than normal starch (70-75% amylopectin, 25-30% amylose). Waxy starches are useful as thickeners or gel-forming agents because they form a clear stable gel with little retrogradation (Morell et al. (1995) Aust. J. Plant Physiol. 22:647-660). High amylose (70%) starch forms insoluble aggregations, is more slowly digested, and therefore is suited for use in formulating adhesives, plastics and as a source of dietary fiber (Whistler R. L. (1984) “Starch” (Eds. R. L. Whistler, J. N. BeMiller and E. F. Paschall) Academic Press: Orlando; Doane (1994) Cereal Foods World 39:556-563). Boyer et al. showed that the increased proportion of larger granules in developing kernels of various genotypes (normal, ae, and ae su) is related to a higher amylose percentage, whereas in late development a decrease in amylose percentage results in a decrease in granule size (Boyer et al. (1976) Cereal Chemistry 53:327-337). The soft starch (h) gene increases starch granule size compared with normal genotypes, and results in a loose packing of starch granules (Gutiérrez et al. (2002) Crop Sci. 42:355-359).
The size and branching patterns of starch molecules also affects physical properties of starch, such as the gelatinization temperature, starch swelling and viscosity. In maize kernels, the sugaryl mutation causes the absence of a debranching enzyme which hydrolyzes alpha-1,6-glycosyl linkages of starch (James et al. (1995) Plant Cell 7:417-429). The mutation results in a decreased concentration of amylopectin and accumulation of the highly branched glucopolysaccharide phytoglycogen, and produces several advantageous physical characteristics of starch. For example, the accumulation of phytoglycogen in sul mutants is associated with smaller and more numerous starch granules (Wang et al. (1993) Cereal Chem. 70:171-179; Brown et al. (1971) Crop Sci. 11:297-302). In addition, starch from sugaryl mutant endosperm containing a high phytoglycogen content has a reduced temperature of gelatinization compared to that of waxy or normal starch (Wang et al. (1992) Cereal Chem. 69:328-334). The reduced gelatinization temperature increases starch solubility after processing (grinding, pelleting, steam flaking) at temperatures below the gelatinization temperature of normal starch. The smaller granule size and reduced temperature of gelatinization may both contribute to the high digestibility of starch from sugaryl mutant corn. See, for example, Fuwa et al. (1979) J. Nutr. Sci. Vitaminol. 25:103-114 and Fuwa et al. (1979) Cereal Chem 54:230-237 and Ninomya et al. (1989) Starch 41:165-167.
Granule size is important in the manufacturing of degradable plastic films, carbonless copy paper, dusting powder, baking powder, laundry-stiffening agents, and utilization of small granules as a fat substitute (Gutiérrez et al. (2002) Crop Sci. 42:355-359). Starch is a major source of carbohydrates for both man and livestock through crops such as wheat, maize, rice and potatoes. Starch is also used industrially in the production of paper, textiles, plastics, adhesives, and provides the raw material for some bioreactors. The type of starch affects the quality of the final product and therefore its profitability. Therefore, methods are needed to modify starch, particularly starch granule size, for use in various industrial applications. One example of an industrial application that may benefit from the use of grain with small starch granules is the corn dry-grind ethanol industry, in which starch of the grain is hydrolyzed to glucose for fermentation into ethanol. Grain with smaller-than-normal starch granules may be hydrolyzed at a lower cost by the α-amylase and glucoamylase enzymes used normally, because smaller starch granules have a larger surface area and would be “attacked” by α-amylase more efficiently (for example, in a quicker fashion, or with reduced amount of enzyme required) than with starch from a normal genetic background.